The actin cytoskeleton is essential for cell motility and depends on the formation of two major actin-based protrusive structures: filopodia and lamellipodia. Both protrusive structures often exist at the leading edge of migrating cells with their formation dependent on the regulation of the barbed-end (fast growing) of actin filaments; however they contribute differentially to cell behavior. Understanding how the cell "determines" which protrusive structures prevails at the leading edge is important to defining the cell's overall migratory behavior and has implication metastasis, development and wound closure. The proposed study focuses on two regulators of the lamellipodia/filopodia transition that bind to the barbed-end of actin filaments, the heterodimeric capping protein (CP) and the Ena/VASP family of molecules. In the proposed study we will: (1) characterize how the various domains of Ena/VASP proteins contribute to filopodia formation using fluorescent and light microscopy, (2) further elucidate CP dynamics using live cell imaging and photobleaching and activation, (3) and investigate how other barbed-end binding proteins contribute to cell motility through depletion by siRNA. [unreadable] [unreadable]